We have shown that the phorbol ester TPA induces GM-CSF mRNA in the murine T cell line EL-4 by stabilizing mRNA. In this study we compared the mechanism of GM-CSF mRNA induction after concanavalin A (ConA) treatment of EL-4 cells to the mechanism of the action of TPA. TPA and ConA both induced GM-CSF mRNA expression transiently and peak mRNA levels were reached within 6 to 10 hours after the addition of either agent. GM-CSF mRNA levels following TPA treatment were higher than those seen with ConA and persisted for 24 hours, whereas GM-CSF mRNA induced ConA decreased significantly after 10 hours. Neither agent caused a significant change in GM-CSF gene transcription rate as determined by nuclear run-on assays. One obvious explanation for the differences in steady-state mRNA levels caused by TPA and ConA treatments could be a difference in GM-CSF mRNA half-life. Half-life (T1/2) measurements were obtained by blocking transcription with actinomycin D and harvesting RNA at various time intervals for Northern analysis. GM-CSF mRNA T1/2 in untreated EL-4 cells is very short (less than 30 min); after TPA treatment GM-CSF mRNA Tl/2 exceeds 3 h; after ConA treatment the Tl/2 is only 1 h. To examine the mechanism of mRNA stabilization, plasmids were constructed containing a reporter gene (chloramphenicol acetyltransferase, CAT) driven by the Rous sarcoma virus LTR. Sequences from the 3' untranslated region (3'UTR) of the GM-CSF gene, including the poly A addition signal, were placed downstream from the CAT coding region in place of the usual SV40 sequences. Constructs were transfected into EL-4 and NIH3T3 cells by electroporation. Transfected cells were left untreated or treated with TPA and analysed for CAT activity. In EL-4 cells transfected with a hybrid RSV/CAT/GM-CSF 3'UTR plasmid CAT activity increased substantially after TPA treatment. A similar increase was not observed in transfected NIH3T3 cells. The data suggest that mRNA stabilization is due to a TPA-inducible factor(s) which is expressed in the EL-4 cells but not in NIH3T3 fibroblasts. Preliminary RNA binding studies indicate the existence of cytoplasmic proteins in T cells which bind to sequences from the 3' UTR of GM-CSF and may mediate message stabilization and/or degradation.